PROJECT SUMMARY: ?Analysis of non-canonical functions of microRNAs? Commitment to cell-fate decisions is fundamental for proper embryonic development and tissue homeostasis. microRNAs, a family of small non-coding RNAs, are among the factors that actively participate in many differentiation processes. Indeed, the last step of differentiation during erythropoiesis is in part governed by miR- 451 and its demise leads to severe anemia. Surprisingly, miR-451 is the only known microRNA whose processing is Dicer-independent but Ago2-dependent. Paradoxically, Dicer is still expressed in erythrocytes, raising the question of what possible advantage represents for erythropoiesis to process miR-451 through a non-canonical pathway. The goal of this project is to uncover by which mechanisms the non-canonical processing of miR-451 becomes indispensable for erythrocyte differentiation. We hypothesize that this alternative processing pathway favors miR-451 production while actively suppressing the Dicer-dependent processing of other microRNAs, thereby ensuring an efficient and precise mechanism to control terminal erythrocyte differentiation. Recent work from our lab and others indicate that miR-451 comprises up to 60% of the microRNA content of maturing erythrocytes, while miR-144 that is co-expressed as a cluster with miR-451 and processed by Dicer only accounts for 1.5%. These results are in striking contrast with our preliminary data that shows that Ago2- dependent processing is not as efficient as Dicer-mediated biogenesis. Surprisingly, our most recent data suggests that pre-miR-451 represses canonical microRNA biogenesis and that Dicer is a target of miR-144. Leveraging all these data, the current proposal examines the hypothesis that miR-451 has a second activity, unrelated to its sequence but dependent on its structural features as a competitive inhibitor of Dicer. Three highly integrated but not interdependent Aims will address the above hypothesis using a set of reprogrammed pre-miR-451 hairpins to i) determine the biochemical features of miR-451/Dicer interaction and their role on canonical miRNA biogenesis, ii) uncover the role of miR-144 in miR-451 processing and iii) identify the erythropoietic processes that most depend on the Ago2-mediated biogenesis of miR-451. Using zebrafish and human iPSC as a model system, the current proposal uses novel genetic and molecular approaches to mechanistically probe the interplay of canonical and non-canonical microRNA processing pathways during erythropoiesis. In doing so, it will uncover mechanisms with the potential to instruct future improvements in blood production form iPSC and therapeutic interventions on anemia. The novelty of the proposal is also driven by an interdisciplinary team that combines experience in microRNAs, zebrafish, bioinformatics and iPSC differentiation. The successful completion of this project will transform our understanding of how microRNAs regulate cell fate and provide invaluable insights to improve iPSC reprogramming to erythrocytes.